CN109874334A - Contact imaging device for fluorescent applications - Google Patents

Abstract

Provide equipment, system, method and the external member for contact imaging.Contact imaging device includes imaging sensor, is mechanically coupled to the fixation fibre faceplate of imaging sensor and is mechanically coupled to the optically filtering layer of fixed fibre faceplate.Optically filtering layer may include interference filter, absorbing filter and/or removable fibre faceplate.Contact imaging device can be used for that fluorescent samples are imaged by filtering out exciting light based on wavelength and/or incidence angle.

Description

Contact imaging device for fluorescent applications

Cross reference to related applications

Present patent application be require on July 17th, 2015 to submit it is entitled " for fluorescent applications contact imaging sets The U.S. Provisional Application of standby (CONTACT IMAGING DEVICES FOR FLUORESCENCE APPLICATIONS) " No.62/193, the non-provisional of 833 priority, is incorporated by reference into this for all purposes.The application is also It is related to the U.S. Patent application of entitled " contact imager (CONTACT IMAGER) " submitted on July 1st, 2015 No.14/789,717, the entire disclosure is incorporated by reference into this for all purposes.

Background technique

The many programs executed in Biochemical Lab are related to the multiple samples or material being distributed on 2 dimensional region Analysis.The example of such program is to each hole for being placed in porous plate (such as 96 hole microtiter plate of standard or bigger plate) In substance, or be applied to the spot (spot) of microscopic size or bigger drop or regular intervals on a solid surface The further example of screening study that carries out of molecular substance be plate shaped running gel, wherein having been carried out parallel multiple The two dimensional electrophoresis of sample separates or one-dimensional separation.Further example is the object being separated by electrophoresis with spot or strips The blotting membrane that matter has been transferred to from slab gel.Skilled biologist will readily appreciate that other examples.

In all these examples, in two-dimensional array the detection and analysis in each site can use it is related to each site The radiation of connection.Detection can simply determine the existence or non-existence of predetermined substance, or can also include based on absolutely basis Or quantitatively determining as the comparison between different loci.In some cases, the radiation detected is by radionuclide decay It causes.In other cases, the radiation detected is light, can be transmitted, absorbs or reflect by each site, or by place Point is generated from the material being in.For example, the substance in running gel or blotting membrane usually passes through fluorescence, chemiluminescence or biology Shine to detect, as the inherent characteristic of the substance at the site, once as substance in entire two-dimensional array quilt Separate the result handled them.The processing may include association reaction, and wherein energy transmitting label is attached to substance, or Substance or label can be irradiated with excitation, this will make them emit the luminous energy most frequently under different wavelength.

In contact imaging, two-dimensional array as discussed above is placed as close to sensor, and uses biography The radiation for the substance being originated from array is imaged in sensor.The example of suitable sensor includes traditional photographic film, non- Crystal silicon (a-Si) sensor and charge (CCD) and complementary metal oxide semiconductor (CMOS) equipment.Sensor Planar or two-dimensional shapes can be configured to, and there is size similar with the size of array that it be used to be imaged.Due to The big region of short distance and exposure between array and sensor, contact imaging allow weak radiation source with high spatial resolution It is detected.

Contact imaging many applications be directed to do not need when detecting the radiation source that energy is entered carried out it is excellent Change.For example, keeping the conventional box of sample array often not influenced by ambient light for photographic film, and array is required to contain There is the light source of endogenous (for example, chemiluminescence).Such box is unsuitable for that fluorescent samples are imaged, and is normally in response to use shortwave The excitation of long light and the light for emitting long wavelength.Because most of fluorogens have the lifetime of excited state of nanosecond order, excitation Fluorogen in array and to be then enclosed in array in dark container to be used for contact imaging be unpractical.

Contact imaging device and program are adapted to fluorescent material and show technological challenge.Sample array must in imaging It must be exposed to exciting light, but the light that mainly must by fluorescence emit of the image of the substance in array rather than exciting light shape At.Contact between array and sensor limits the selection for preventing exciting light from reaching sensor.

Summary of the invention

It provided herein is the equipment being imaged for contact, system, method and external members.

In the first aspect of the invention, a kind of contact imaging device is provided.The contact imaging device includes into As sensor, it is mechanical coupling to the fixation fibre faceplate of imaging sensor and is mechanical coupling to the optically filtering of fixed fibre faceplate Layer.In some embodiments, the numerical aperture that fixed fibre faceplate has is at most about 0.1,0.2,0.3,0.4,0.5,0.6, 0.7,0.8,0.9,1.0, or increment or gradient in the range of about 0.1 to 1.0.

In some embodiments of contact imaging device, optically filtering layer includes interference filter.Interference filter can To be long pass filter, with block ultraviolet and/or visible light can be made to pass through.For example, interference filter can make to have 400-550,400-500,400-450,400-425,425-450,450-475,475-500,500-525 or 525-550nm model The light of wavelength in enclosing passes through.In these embodiments, other than interference filter, optically filtering layer be may further include Absorbing filter.Absorbing filter may include coloured glass, and/or be arranged on interference filter and fixed fibre faceplate it Between.

In some embodiments, the optically filtering layer of contact imaging device includes absorbing filter.Absorbing filter can To include coloured glass.In these embodiments, for example, fixed fibre faceplate can have at most about 0.1,0.2,0.3,0.4, 0.5,0.6,0.7,0.8,0.9,1.0, or the numerical aperture of increment or gradient in the range of about 0.1 to 1.0.Alternatively or Additionally, fixed fibre faceplate may include multiple optical fiber, and the core of at least one optical fiber in optical fiber or covering can wrap Include absorbent material.

In some embodiments of contact imaging device, optically filtering layer includes that fibre faceplate and optically filtering can be removed Device, removable fibre faceplate are applied on the side of optical filter, and optically filtering layer is configured as from fixed optical fiber Panel is removed.For example, transmission filter can be interference filter or absorbing filter.The numerical aperture of fibre faceplate can be removed Diameter can be such as at most about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0, and/or can be less than and fix Fibre faceplate numerical aperture (for example, small at least about 0.01,0.02,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7, 0.8,0.9,0.95 or 0.99).Removable fibre faceplate can be set between fixed fibre faceplate and optical filter.

In some embodiments, transmission filter setting is between fixed fibre faceplate and removable fibre faceplate.At this In a little embodiments, transmission filter can be contacted with fixed fibre faceplate, and/or can be interference filter.Optical fiber can be removed Panel can have the numerical aperture of such as at most about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0, and/ Or fixed fibre faceplate can have for example, at least about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,0.95 or 0.99 numerical aperture.

In some embodiments of contact imaging device, fixed fibre faceplate is optical fiber cone.In some embodiments, Fixed fibre faceplate includes multiple optical fiber, and the core of at least one optical fiber in optical fiber or covering include absorbent material.Example Such as, the core of at least one optical fiber, the core or covering of the covering of at least one optical fiber and/or all optical fiber may include absorbability material Material.

In some embodiments, for example, the acceptance angle of the optical fiber of fixed fibre faceplate be most about 1,2,3,4,5,6,7, 8,9,10,20,30,40,50,60,70,80 or 90 degree.In some embodiments, fixed fibre faceplate includes being dispersed in multiple light Multiple black optical fiber among fibre.Black optical fiber can have the cross section of circular cross section or hexagon, and/or setting exists In clearance space between optical fiber.The quantity of black optical fiber and the ratio of number of optical fiber in fixed fibre faceplate can be (example As) at most about 1:1,000,1:500,1:200,1:100,1:50,1:20,1:10,1:5,1:2,1:1,2:1,5:1,10:1, 20:1,50:1,100:1,200:1,500:1 or 1,000:1 and/or black optical fiber can take up the transversal of fixed fibre faceplate Face area (for example) at most about 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, 90%, 95% or 99%.

In the second aspect of the invention, provide for fluorescence, phosphorescence or chemiluminescence sample to be imaged System.The system includes excitation light source and above-mentioned contact imaging device.In some embodiments, which further comprises Fluorescence, phosphorescence or chemiluminescence sample.In some embodiments, which further comprises box (cassette), wherein should Box includes substrate (base plate) and lighttight lid, and the contact imaging device is mechanical coupling to the base Plate.Substrate may include heat-conducting metal plate (slab), and imaging sensor can be coupled to metal plate.In some realities Apply in example, which further comprises excitation fiber panel, wherein excitation fiber panel be configured as setting in excitation light source and Between sample.Excitation fiber panel can be mechanical coupling to excitation light source.Excitation fiber panel can have for example at most about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0 numerical aperture.Emitted by excitation light source and by excitation fiber The light of plane transport can be collimated.

In some embodiments of the system, the collimated light of excitation light source transmitting.In some embodiments, excitation light source It is positioned such that the light emitted by excitation light source is not incident on optically filtering layer relative to contact imaging device.Some In embodiment, excitation light source is positioned such that the light emitted by excitation light source with extremely from normal relative to contact imaging device Few about (for example) 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80 or 90 degree of angle is incident on optically filtering On layer.In some embodiments, excitation light source is positioned such that excitation light source is being parallel to relative to contact imaging device Emit light on the direction on the surface of optically filtering layer.

Any embodiment of the system, which is configured such that, passes through sample by the light of excitation light source transmitting.

In the third aspect of the invention, the side for fluorescence, phosphorescence or chemiluminescence sample to be imaged is provided Method.This method includes being positioned to sample close to above-mentioned contact imaging device, exposes the samples to excitation light source, and make The image of sample is obtained with the imaging sensor of contact imaging device.

In some embodiments of this method, exposed sample and the image for obtaining sample occur simultaneously.In some embodiments In, the image of sample is obtained using time-resolved fluorescent technique.In some embodiments, before the image for obtaining sample, Excitation light source is exposed the samples to, excitation light source is then shut off.In some embodiments, sample is phosphorescent.This method Some embodiments further include the image that Image Sharpening Algorithm is applied to sample.

In the fourth aspect of the invention, a kind of external member is provided.The external member includes being mechanical coupling to fixed fibre faceplate Imaging sensor, the first optically filtering layer and the second optically filtering layer, wherein each optically filtering layer includes that optical fiber can be removed Panel and optical filter simultaneously are configured as being mechanical coupling to fixed fibre faceplate, and removable fibre faceplate is applied to and optics On the side of filter.

In some embodiments of the external member, the transmission filter of the first optically filtering layer is interference filter, and the The transmission filter of two optically filtering layers is absorbing filter.In some embodiments, the first optically filtering layer and the second optics The optical filter of wave filtering layer makes to pass through in the light of different wave length band.In some embodiments, the first optically filtering layer and second The removable fibre faceplate of optically filtering layer has different numerical apertures.In some embodiments, the first optically filtering layer quilt It is configured to for the removable fibre faceplate being arranged between the fixed fibre faceplate and the optical filter, and described Second optically filtering layer is configured as the transmission filter being arranged in the fixed fibre faceplate and removable fibre faceplate Between.

Detailed description of the invention

Illustrative aspect and embodiment are described in detail below with reference to the following drawings.

Fig. 1 shows contact imaging device 100 according to some embodiments of the present invention.Imaging sensor 101 is mechanical It is coupled to fixed fibre faceplate 102, fixed fibre faceplate 102 is mechanical coupling to interference filter 103 again.

Fig. 2 shows contact imaging devices 200 according to some embodiments of the present invention.Imaging sensor 201 is mechanical It is coupled to fixed fibre faceplate 202, fixed fibre faceplate 102 is mechanical coupling to optically filtering layer again.Optically filtering layer includes inhaling Receive filter 203 and interference filter 204.Interference filter be placed on absorbing filter with fixed fibre faceplate and imaging The opposite side of sensor.

Fig. 3 shows contact imaging device 300 according to some embodiments of the present invention.Imaging sensor 301 is mechanical Ground is coupled to fixed fibre faceplate 302, and fixed fibre faceplate 302 is mechanical coupling to optically filtering layer 303 again.Optically filtering layer packet Include the transmission filter 305 on removable fibre faceplate 304 and side coated in removable fibre faceplate.Fiber facet can be removed Plate and fixed fibre faceplate one are stacked on top of the other, so that being transmitted the light of the transmission of filter 305 can be guided To imaging sensor 301.

Fig. 4 shows contact imaging device 400 according to some embodiments of the present invention.Imaging sensor 401 is mechanical Ground is coupled to fixed fibre faceplate 402, and fixed fibre faceplate 402 is mechanical coupling to optically filtering layer 403 again.Optically filtering layer packet Include the interference filter 404 being applied on the side of removable fibre faceplate 405.As depicted, interference filter is set It sets between fixed fibre faceplate and removable fibre faceplate.The numerical aperture that fibre faceplate can be removed can be lower than fixed optical fiber The numerical aperture of panel.

Fig. 5 shows the fibre faceplate 500 with optical fiber light core 501 and covering 502.According to the aspect of the disclosure, covering Absorbent material including such as coloured glass or black glass etc.

Fig. 6 shows the optical fiber light core for having optics covering 603 and black covering 602 of the aspect according to the disclosure 601 fibre faceplate 600.

Fig. 7 shows the optical fiber with the black optical fiber 702 in the clearance space between the composition optical fiber that panel is arranged in Panel 700.According to the aspect of the disclosure, optical fiber includes core 701 and covering 703.

Fig. 8 A shows having with every 61 total optical fiber one in fibre faceplate 800 according to the aspect of the disclosure The ratio of black optical fiber 802 is combined the optical fiber light core 801 of setting and the fibre faceplate 800 of covering 803 with black optical fiber 802.

Fig. 8 B shows having with every 61 total optical fiber three in fibre faceplate 800' according to the aspect of the disclosure The ratio of root black optical fiber 803 is combined the optical fiber light core 801 of setting and the fibre faceplate 800' of covering 803 with black optical fiber 802.

Fig. 8 C show according to the aspect of the disclosure have in the array of total optical fiber for being distributed in fibre faceplate 800 " Black optical fiber 802 combine setting optical fiber light core 801 and covering 803 fibre faceplate 800 ".

Fig. 9 A shows the system 900 for fluorescent samples to be imaged according to some embodiments of the present invention.It connects Touch imaging device includes imaging sensor 901, fixed fibre faceplate 902 and optically filtering layer 903.Optically filtering layer 903 wraps Include the optical filter of such as interference filter or absorbing filter etc.Two-dimentional sample array 904 is placed with to be filtered with optics Wave layer 903 is contacted and is substantially laid flat.Excitation fiber panel 905 is arranged between excitation light source 906 and sample array 904.Cause This, the light 907 emitted by excitation light source 906 is collimated, and light 908 hits sample with low incidence angle or close to zero incidence angle Product array 904 and optically filtering layer 903.

Fig. 9 B shows the system 900 ' for fluorescent samples to be imaged according to some embodiments of the present invention.It connects Touch imaging device includes imaging sensor 901 and fixed fibre faceplate 902.Excitation fiber panel 905 is arranged on exciting light Between source 906 and fixed fibre faceplate 902.Alternative optically filtering layer 910 includes such as interference filter, dielectric or suction Receipts filter etc is arranged on excitation fiber panel 905 and towards the optical filter for fixing fibre faceplate 902.Absorb glass Glass 909 concatenates ground (in series) with fixed fibre faceplate 902 and places.Therefore, the light 907 emitted by excitation light source 906 is quasi- Directly, and at least partly it is filtered, and light hits fixed fibre faceplate 902 with low incidence angle or close to zero incidence angle. Also show (or the sample of sample gel 911 for contacting with excitation fiber panel 905 and substantially laying flat irradiated by light source 906 Trace (blot)).

Fig. 9 C show further diagram light source according to some embodiments of the present invention in figures 9 b and 9 shown in be The exploded view of system 900'.Light source 906 is positioned on the side of sample gel 911, so that optical transport passes through 911 court of sample gel To and by excitation fiber panel 905, heat absorbing glass 909 and fibre faceplate 902 is fixed to sensor 901.Light source 906 can be by Referred to as total internal reflection (TIR) structure, including having the LED structure 912 of one or more color LEDs as primary illumination source. In some respects, LED structure 912 can have one or more groups of colored or white light LEDs.In further, LED can be with With the filter being placed between each LED and transparent (clear) TIR layer 914.TIR structure may include (far from sample Gel 911 arrives order most nearby) reflecting layer 913, transparent TIR layer 914, diffusing globe 915 and secondary hyaline layer 916.LED 912 can be positioned so that and be radiated on the side of transparent TIR layer 914, and wherein optical transport passes through transparent TIR layer 914.Reflecting layer 913 lead back to light incident thereon towards sample gel 911.Diffusing globe 915 can be transmitted in light towards sample gel 911 When by light uniformization.Secondary hyaline layer 916 can provide physics when light is passed to sample gel 911 from light source for TIR structure Limitation.Side reflector 917 can be positioned on the side opposite with TIR structure of LED structure 912, by light towards TIR structure It reboots back.In a further embodiment, light source 906 can be located at the side of TIR structure, the two sides of TIR structure, TIR On three sides of structure or all sides of TIR structure.

Figure 10 shows the system 1000 for fluorescent samples to be imaged according to some embodiments of the present invention.It connects Touch imaging device includes imaging sensor 1001, fixed fibre faceplate 1002 and optically filtering layer 1003.Optically filtering layer 1003 include the optical filter of such as interference filter or absorbing filter etc.Two-dimentional sample array 1004 be placed with Optically filtering layer 1003 is contacted and is substantially laid flat.Light source 1005 is positioned to the side of contact imaging device, so that light 1006 surfaces for being nearly parallel to optically filtering layer are advanced, and hit sample array 1004 with high incidence angle.In some cases Under, these light pass through sample array before exciting interested analyte.

Figure 11 shows contact imaging device 1100 according to some embodiments of the present invention.1101 machine of imaging sensor Tool is coupled to fixed fibre faceplate 1102, and fixed fibre faceplate 102 is mechanical coupling to optically filtering layer 1103 again.Optically filtering layer Including the interference filter 1104 being coated on the side that fibre faceplate 1105 can be removed.Interference filter is arranged on fixed light Between fine panel and removable fibre faceplate.Thick sample array 1106 contacts removable fibre faceplate 1105 and can be sudden and violent It is exposed to excitation light source (not shown).Fibre faceplate, which can be removed, has low numerical aperture, and for point in sample array 1106 Analysis object carries out imaging and provides the high depth of field.

Specific embodiment

A. it introduces

Inventor has found in conjunction with the equipment of fibre faceplate and optical filter suitable for fluorescent material or sample Contact imaging.Equipment includes one or more fibre faceplates and one or more optical filters, these fibre faceplates and light Filter is mechanically coupled to each other and is placed on the sample side of imaging sensor.Such equipment, which can be configured as, to be made by sample The light emitted to substance fluorescence in product passes through, while stopping exciting light.The equipment can be based on wavelength and the close sensing of light The angle of device selects light.There is provided herein equipment, system, method and the external members for executing contact imaging.

B. it defines

Unless otherwise defined, otherwise terminology used herein and scientific term have and those of ordinary skill in the art Normally understood identical meaning.See, e.g. " Cytobiology and molecular biology dictionary " (DICTIONARY OF of Lackie CELL AND MOLECULAR BIOLOGY), Elsevier (Elsevier) (the 4th edition, 2007)；" the molecule of Sambrook etc. Clone, laboratory manual " (MOLECULAR CLONING, A LABORATORY MANUAL), Cold Spring Harbor Publications (Cold Springs Harbor Press) (Cold SpringHarbor (Cold Springs Harbor), NY 1989).It provides defined below to help Understand the certain terms frequently used herein, and does not mean that and limit the scope of the present disclosure.

Term " film " includes trace and film (for example, nitrocellulose, nylon Kynoar (PVDF) and commonly used in the art Other materials).Film is also possible to paper or paper mixture, for example, transfer and the drying on paper that wherein measurement is related to gel. " sample film " refers to for example from gel transfer or the film of direct applied carrying sample.

" analyte " refers to the molecule or molecular complex that can be detected or be imaged as provided herein.Analyte can To be biological source or can be synthesis.Analyte may include peptide, protein, nucleic acid, carbohydrate, lipid, disease Poison, metabolin, hormone, confactor, vitamin, drug and/or small molecule.Without restriction, analyte can be it is polar, It is electrification, hydrophilic, hydrophobic, monomer, oligomer or polymerization, and can have any molecular weight.

Term " sample " or " biological sample " cover the various sample types obtained from organism.The term covers such as blood Liquid, blood constituent, saliva, serum, blood plasma, the body fluid of other fluid samples etc of urine and biological source, solid tissue are living Inspection, tissue culture or the supernatant for being derived from culture cell.Biological sample processed can for example remove thin before measurement Born of the same parents or cell fragment.The term is covered after they are obtained such as by being handled, being dissolved with reagent, settled or to certain Component is enriched with the sample operated.Sample may include analyte.

" sample array " refers to the physical matrix that the analyte of wherein one or more samples is separated and/or is distributed. Some sample array are two-dimensional, because they include the analysis from a sample being distributed in two or more dimensions Object, or comprising the analyte from multiple samples, wherein the analyte from each sample is distributed on one or more dimensions On degree.The example of sample array is porous plate, microtiter plate, plate shaped running gel and blotting membrane.

As used herein, " immunoassays " refer to the measurement dependent on antibody-antigene interaction.Example includes protein Trace (is transferred to the antigen of film, is exposed to the film of antibody, and in some embodiments, secondary antibody)；ELISA；With other bases In the marker determination of affinity.

Term " antibody " as used herein refers to the polypeptide of specific binding and discriminance analysis object (antigen).The term is contained Lid is anti-by immunoglobulin gene or multiple immunoglobulin genes, its recombination and/or clone variants and its fragment coding Body.Full length antibody is the tetramer.Each such tetramer is made of two pairs of identical polypeptide chains, each pair of to have " light " chain (about 25kD) and " weight " chain (about 50-70kD).The N-terminal of every chain, which defines, is mainly responsible for about 100 of antigen recognizing extremely The variable region of 110 or more amino acid.Term " variable light " (VL) and " variable heavy chain " (VH) respectively refer to these light chains And heavy chain.Generally acknowledged light chain immunoglobulin is classified as κ or λ.Heavy chain immunoglobulin is classified as γ, μ, α, δ or ε, Define immunoglobulin class IgG, IgM, IgA, IgD and IgE respectively again.

The terms such as term " right ... to have specificity ", " specific binding " refer to at least 2 times bigger than non-targeted compound Affinity (for example, at least big 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times or 100 times appoint What one affinity) molecule (for example, antibody or antibody fragment) in conjunction with its target.For example, specific binding is given anti- The antibody of body target would generally be with 2 times at least bigger than non-antibody target of affinity binding antibody target.Standard side can be used Method determines specificity, such as solid phase ELISA immunoassays (write " real using antibody see, e.g. Harlow and Lane Test room handbook (1998) " (Using Antibodies, A Laboratory Manual (1998)), which depict about available In the form and condition of the immunoassays for determining specific immunoreactivity).

When about antibody target (for example, antigen, analyte) by use, term " in conjunction with " be indicated generally at antibody combine it is pure Most of antibody target (assuming that molar ratio appropriate) in group.For example, the antibody of given antibody target is combined usually to combine At least 2/3 in solution (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, antibody target any of 98%, 99% or 100%).Technical staff, which will be recognized, to be depended on determining the side combined Method and/or threshold value will appear some changeabilities.

The terms such as term " label ", " detectable label ", " detectable part " refer to can be by spectroscopy, photochemistry, life Object chemistry, immunochemistry, chemistry or the composition of other physical means detection.In the context of the disclosure, label is generally included Luminescence reagent, electron-dense reagents, enzyme (for example, such as in ELISA common), biotin, digoxin,³²P and other same positions Element, haptens and for example it can be checked or be used for detection by the way that radioactive label to be incorporated in peptide and react with peptide specific Antibody protein.The term includes the combination of single labelled reagent, for example, for example mentioning under specific wavelength or wavelength combination For the dye combinations of unique detectable feature.It can be used known in the art for that will mark and the coupling of desired reagent (conjugate) any method, such as use " the bioconjugate technique " (Bioconjugate write in Hermanson Techniques) 1996, Academic Press, Inc (Academic Press, Inc.), description in Santiago (San Diego) Method.

When mentioning result or signal, term " positive (positive) " instruction presence is being detected in the sample Analyte or article.When mentioning result or signal, term " negative (negative) " instruction there is no in the sample by The analyte or article detected.Positive and feminine gender at least one usually by compareing (for example, being confirmed as sun for sample Threshold level needed for property) or negative control (for example, as it is known that blank) be compared to determine.

" control (control) " sample or value refer to be used for as reference (commonly known reference) and test sample into The sample that row compares.For example, test sample can be acquired from test condition (such as in the case where there is test compound), and with From known conditions, (such as under there is no test compound (negative control), or there are (positive right under known compound According to)) sample be compared.Control can also indicate the average value collected from a large amount of tests or result.The technology of this field Which personnel will be understood that impinging upon given feelings it will be recognized that control can be designed to assess any amount of parameter It is valuable under condition, and data can be analyzed based on compared with control value.Compare the conspicuousness for determining data It is also valuable.For example, if the value of given parameters be in control it is variable, the variation in test sample will not be recognized To be significant.

If term is used herein, if two objects are in contact with each other, and an object is relative to another object The movement of body is such as suffered restraints or is hindered by friction, then the two objects are by " mechanical couplings ".The constraint or obstruction of movement Amount can be designed to the desired application of two objects.For example, the machinery between imaging sensor and fibre faceplate In coupling, fibre faceplate can be maintained on position, be allowed to using propagating through panel and to the light on sensor Consistent image is obtained during the desired time.The object of mechanical couplings can reasonable time using tool, power or Other modes to be separated.

Under the context for the light being incident on surface, term " incidence angle " has a conventional meaning, i.e., light with it is vertical Angle between the line on surface.

Term " acceptance angle " has conventional meaning, that is, light can be close to the end of optical fiber and be transmitted through light Fine maximum incident angle.When being applied to fibre faceplate, which refers to the acceptance angle for constituting the optical fiber of fibre faceplate.

Term " numerical aperture " (being abbreviated as " NA ") has conventional meaning, and is to characterize such as lens or optical fiber optics The dimensionless number of the angular range of the acceptable incident light of the optical device of device etc.In some cases, for optical fiber light Device is learned, when being measured in air, NA=n sin (θ), wherein n is the refractive index of working media, and θ is acceptance angle.

Term " black " refers to the material for substantially absorbing all wavelengths of visible light, so that these materials exhibit black. In some cases, black material does not transmit and/or does not reflect visible light substantially, or transmission and/or reflectivity they inhale That receives is few (for example, few 1.5,2,5,10,20,50,100,200,500,1000,100,000,1,000,000 or 10,000,000 Visible light again).Deciding grade and level be ND2, ND4, ND8, ND16, ND32, ND64, ND100, ND128, ND256, ND512, ND1000, ND1024 or higher neutral density filters are the examples of the optical device containing black material.

Term " about " and " approximately equal " be used to modify numerical value and indicate the restriction range near the value herein. If " X " is the value, " about X " or " being approximately equal to X " is indicated generally at the value from 0.90X to 1.10X.To any ginseng of " about X " Examine instruction at least value X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X and 1.10X.Therefore, " about X " is intended to public It creates a precedent such as " 0.98X ".At the beginning of " about " is applied to numberical range, it is suitable for the both ends of range.Therefore, " from about 6 to 8.5 " are equivalent to " from about 6 to about 8.5 ".When " about " being applied to first value of a class value, it is suitable for that group All values.Therefore, " about 7%, 9% or 11% " is equivalent to " about 7%, about 9% or about 11% ".

C. sensor

Any convenient and fast imaging sensor can be included in this equipment.The example of suitable imaging sensor is complementary Metal-oxide semiconductor (MOS) (CMOS) sensor or charge (CCD) sensor.Such sensor and application method Be in the art it is well known, for example, Fraden (Springer, the 4th edition) modern sensor handbook: physics is designed and is answered With (Handbook of Modern Sensors:Physics, Designs, and Applications)；Cabello etc. (2007)Phys Med Biol 52:4993.Useful sensor can be obtained from many commercial suppliers, for example, Canon, Samsung, Toshiba, cmos sensor etc..Instead or in addition, imaging sensor may include photographic film.

Both CCD and cmos sensor have the two-dimensional array of thousands of or millions of a minute cells, each of these Signal (light or radiation) is all converted into electronics from a fraction of image.Once image is captured on a sensor, next Step is exactly to read the value of each unit in image (charge of accumulation).In CCD equipment, charge is transported across chip and in array An angle on be read.The value of each pixel is converted to digital value by analog-digital converter.In most of CMOS devices, every There are several transistors at a pixel, is amplified using more conventional electric wire and dislocation charge.CMOS method is more flexible, because Each pixel can be individually read.Ccd sensor uses special manufacturing process, and charge is allowed to be transported and do not had across chip There is distortion.This process leads to the sensor of very high quality in terms of fidelity and light sensitivity.

Current CCD and cmos imaging system are (for example, C-DiGit blot scanners, Dexela and myECL imager system System) it is limited to generate digital picture.Presently described equipment with similar imaging technique and software can be used in system.Below The box of description can be easily connected to computer or scanner during exposure or after exposure, to detect depositing for signal , be not present and/or intensity.

Control panel can be used to obtain in signal from sensor, the control panel can for example be placed with in equipment Sensor it is adjacent, or outside can be placed on.Therefore, sensor can use cable or by port or wireless connection quilt It is connected to plate.Control panel then can be with the external device communication of offer user interface.External equipment may include touch screen, place Manage device or storage equipment.The example of external equipment includes handheld device (for example, smart phone or tablet computer), meter on knee Calculation machine and desktop computer.External equipment can by cable on the box of fenced contact imaging device or port (for example, USB or Ethernet) or accessed by wireless signal (for example, WiFi or bluetooth).

In some embodiments, imaging sensor or entire contact imaging device can be cooled during exposure.It is colder Temperature reduce the dark current (potential source of noise) in digital sensor and realize longer time for exposure, can be improved Sensitivity.However, cooling slows down really, there may be the chemical reaction of optical signalling (for example, chemiluminescence or fluorescence).User Cooling imaging sensor can be determined whether based on the considerations of speed and sensitivity.

D. equipment

Contact imaging device according to an embodiment of the present invention includes imaging sensor as disclosed above and mechanical couplings To the fixation fibre faceplate of imaging sensor.Fibre faceplate (also referred to as optical fiber optic panel, optical fiber optical sheet (FOP), fibre optic plate Or referred to as panel) be short fiber coherence beam.There is the coherence beam and fibre faceplate quilt of multiple short fibers in fibre faceplate In the case where being optically coupled to sensor, it is intended to provide the optical fiber of sufficient density of each coherence beam, so that relevant from one The optical fiber of beam focuses on desired sensor element, and will not sensor adjacent or near to sensor element in light Learn ground " scatter (bleed over) " or creation visual noise.In some respects, fixed fibre faceplate can be for example including at least About 2,5,10,20,50,100,200,500 or 1,000,2,000,5,000 or 10,000 optical fiber, in this paper, it is also referred to as " composition optical fiber ".In other respects, the quantity for forming optical fiber can be according to each sensor element or each sensor pixel Density determines.In such aspect, the ratio of the composition optical fiber of each sensor pixel can be 2:1,5:1,10:1, 15:1,20:1,80:1,100:1,200:1,500:1,1,000:1,10,000:1, or the ratio increment within the scope of this. In some embodiments, sensor pixel can be for from about 5 microns to about 200 in surface size (for example, width, diameter) Micron (5 μm -200 μm), or it is in its increment or gradient.In a specific embodiment, sensor pixel can be about 80 Micron (80 μm) or about 150 microns (150 μm).

Each optical fiber includes core and covering, and can by total internal reflection from the side of fibre faceplate to the other side along Its length transmission light.In some embodiments, optical fiber is parallel to each other.Fibre faceplate (can be looked into from Incom Corp. (InCom, Inc.) Er Dun (Charlton), Massachusetts, the U.S.), ProxiVision limited liability company (GmbH) (Ben Sihaimu (Bensheim), German), collimating aperture company (Collimated Holes, Inc.) (Campbell (Campbell), Jia Lifuni Sub- state, the U.S.), fibre system company (Fiberoptic Systems, Inc.) (Simi Valley city (Simi Valley), Jia Lifu Buddhist nun Asia state, the U.S.), edmond optics (Edmund Optics) (Bahrain (Barrington), New Jersey, the U.S.) etc. It is commercially available.Fibre faceplate can be by glass (for example, low-melting glass, such as borosilicate and lead base glass) or by such as It is prepared by the organic polymer of polycarbonate and polystyrene etc.

In some embodiments, fibre faceplate is plane on one or two surface of composition optical fiber termination, and And generally there is flat shape.Therefore, fibre faceplate can abut directly against planar imaging sensor, and can will sense Device is separated with two-dimentional sample array (for example, running gel or blotting membrane), while meeting the shape of sample array.In some implementations In example, fibre faceplate is positioned such that form fiber perpendicular is orientated in imaging sensor and/or sample array.In some implementations In example, fibre faceplate is coupled to sensor, so that composition optical fiber is specifically aligned with the pixel of sensor.For example, each light Fibre can terminate at one or more pixels or each pixel can be across the cross section of one or more optical fiber.It is any Available mechanism can be used to fibre faceplate being coupled to imaging sensor.It is, for example, possible to use screw or transparent adhesive tape by it Be coupled.In the embodiment that imaging sensor is photographic film, film can be inserted into frame, the frame and quilt It is coupled to fibre faceplate, a piece of new film is allowed to be used to expose every time.If desired, can be by protectiveness or non-anti- The one or more surfaces for applying the coating to fibre faceplate of penetrating property.

Light can save image by the transmission of fibre faceplate from a surface of fibre faceplate to next surface.Cause Thicker fibre faceplate can be used when using transmitted light to form image to replace glass or plastics comparable to obtain in this Quality image.Fixed used in some embodiments of this contact imaging device fibre faceplate at least about 0.1mm, The thickness of 0.2mm, 0.5mm, 1mm, 2mm, 5mm, 10mm, 20mm or 50mm or its increment and gradient.Thicker fibre faceplate can More mechanical supports are provided with the imaging sensor and/or two-dimentional sample array being imaged that are coupled to it.Further, The thickness of fixed fibre faceplate can provide the thermistor for maintaining temperature.In further embodiment, fixed light Fine panel can have the structure of taper, partly focus or concentrate optical path, wherein fixed fibre faceplate can have thickness 10mm, 20mm, 50mm or 100mm or its increment and gradient.

Some embodiments of contact imaging device include being designed to permit the fixation optical fiber that light enters with low incidence angle Panel.In these embodiments, panel can be transmitted and (be received) to be approximately perpendicular to the close light of the angle of panel surface, and And absorb, reflect or otherwise stop (that is, refusal) close with higher incidence angle (for example, being roughly parallel to panel surface) Light.Alternatively or equally, with the long axis of these optical fiber forming close to panel substantially in parallel (that is, with small incidence angle) The light of optical fiber can be received, and the light for being approximately perpendicular to fiber axis (that is, with big incidence angle) can be rejected.In some implementations In example, fixed fibre faceplate has low numerical aperture, such as most about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 Or 1.0.In some embodiments, the acceptance angle of the optical fiber of fixed fibre faceplate be most about 1,2,3,4,5,6,7,8,9,10, 20,30,40,50,60,70,80 or 90 degree.Fibre faceplate with low numerical aperture and/or acceptance angle can be used on the one hand Slave sample array in substance fluorescence between the exciting light of the light emitted and another aspect being originated from outside sample array into Row is distinguished.For example, panel can receive and transmit the light for being passed directly to imaging sensor from sample array, and refuse (blocking) Light from sample array periphery.Low numerical aperture and/or acceptance angle can also increase the depth of field of fibre faceplate, this can be for To thick sample array, or separated by one or more material layer and fibre faceplate and imaging sensors between It is useful that sample array, which carries out imaging,.

Contact imaging device further includes the optically filtering layer for being mechanical coupling to fixed fibre faceplate.Optically filtering layer can be with By make the wavelength of interested light by and stop other wavelength and be based on wavelength filtering light.Any kind of filter can be with It is used in optically filtering layer, such as absorbing filter, interference filter, dichroic filter, long pass filter, short pass filter Device, bandpass filter, bandstop filter, more band filters, broadband filter or neutral density filters.Optically filtering layer can be with Fixed fibre faceplate for example is mechanically coupled to using adhesive or fastener according to expectation.

In some embodiments, optically filtering layer is located on the side opposite with imaging sensor of fixed fibre faceplate, So that optically filtering layer, fixed fibre faceplate and imaging sensor one stack on top of the other.Therefore, fixed fibre faceplate It can be clipped between imaging sensor and optically filtering layer, and receive and passed through the light that optically filtering layer is filtered.As at As sensor and fixed fibre faceplate, optically filtering layer can be flat, plane and/or it is mutual with sample array in shape It mends.Optically filtering layer can be configured as directly contact sample array.

In some embodiments, optically filtering layer includes interference filter (Fig. 1).Interference filter generally includes to have not With the compound glass of refractive index, plastics or other dielectric materials.Interface between, the layers depends on incidence angle, wavelength And other factors, light can be transmitted or be reflected.Interference filter (otherwise referred to as dichroic filter or film filter) Can from Edmund Optics (Barrington, New Jersey, USA) and Omega Optical (Brattleboro, Vermont, USA) etc. it is commercially-available.In some embodiments, interference filter is long pass filter, such as by being greater than The wavelength of the light of 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm or 850nm or its increasing Amount, range and gradient.In some embodiments, interference filter block ultraviolet and/or pass through visible light.Interference filter can To be configured as the wavelength by the light by the chemiluminescent substrate or Fluorophore emission that typically use.For example, filter can Make to have in about 400-550nm, 400-500nm, 400-450nm, 400-425nm, 425-450nm, 450-475nm, 475- The light of wavelength within the scope of 500nm, 500-525nm or 525-550nm passes through.Interference filter can be additionally configured to stop normal Insight is used to excite the wavelength of the light of fluorogen, such as the wavelength within the scope of near ultraviolet, has the wave from about 350nm to 400nm Long light or the chemically light of light emitting source transmitting.

Instead of interference filter or other than interference filter, optically filtering layer may include absorbing filter.It absorbs The light absorbing certain wavelength of filter simultaneously transmit other wavelength.Some absorbing filters contain organic dyestuff or pigment or other Coloured organic or inorganic material.The example of absorbing filter includes coloured glass filter and plastics (for example, polycarbonate or third Olefin(e) acid) filter.Absorbing filter can (that gulf city (Irvine), adds benefit from Li Bo company (Newport Corporation) The state Fu Niya, the U.S.), Soret wins company (Thorlabs, Inc.) (newton city (Newton), New Jersey, the U.S.) He Xiaote Group (Schott AG) (Mainz (Mainz), Germany) and other sources obtain.

As incidence angle increases, absorbing filter usually transmits progressively less light, and interference filter transmission is more Light.Therefore, it is transferred to the angle and wavelength of the light of the fixation fibre faceplate and imaging sensor in this equipment for control, inhales Receiving filter and interference filter can be replaced by another or be applied in combination.In some embodiments, optically filtering layer includes inhaling Receive both filter and interference filter.For example, interference filter can be relative to fixed fibre faceplate and imaging sensor quilt It is placed on before absorbing filter, so that absorbing filter is arranged on (figure between interference filter and fixed fibre faceplate 2).Therefore, the light from sample array passes through interference filter first, then passes through absorbing filter.In this configuration, no The light of desired wavelength can be filtered out step by step, and the light at this low incidence angle is filtered out by interference filter, and high incidence angle Light be absorbed filter and filter out.Alternatively, it can filter in reverse order, interference filter setting is absorbing filtering Between device and fixed fibre faceplate.Interference filter and absorbing filter for those skilled in the art, in optically filtering layer Various configurations will be apparent.

For the flexibility to various sample array and the imaging of substance that they are included, optically filtering layer can be can It removes.For example, optically filtering layer may be configured to be attached mechanically to fixed fibre faceplate, and then when appropriate Between be detached from.Reversible mechanism can be used for being attached and be detached from, such as screw, guide rail or clip.It (is discussed below in some embodiments ) in, contact imaging device is provided with the external member of two or more optically filtering layers, each optically filtering layer tool There is the configuration of different optical components.Any one of these layers can be coupled to fixed fibre faceplate, and optics is filtered Wave layer can jointly be used for different wave-length coverages (for example, for the excitation of the substance in sample array or by these The transmitting of substance), different sample array characteristic (for example, thickness or ingredient) and other contingent conditions for imaging.

In some embodiments, optically filtering layer includes that fibre faceplate and transmission filter can be removed (for example, interference filter Wave device or absorbing filter).It is similar with fixed fibre faceplate that fibre faceplate can be removed, and the coherence beam including short fiber.By In the guide-lighting property of fibre faceplate, two or more fibre faceplates can be stacked, and can be from being projected through The light of stacking forms image.The image can have resolution ratio equal or comparable with the image formed by single fibre faceplate, Because of little or no light loss or diffusion when passing through between fibre faceplate in a stack.In some embodiments, optics Wave filtering layer is configured such that removable fibre faceplate directly contacts fixation fibre faceplate and/or removable fibre faceplate and consolidates It is parallel for determining the composition optical fiber of fibre faceplate.The example of such embodiment is shown in FIG. 3, wherein fibre faceplate can be removed 304 can be arranged between fixed fibre faceplate 302 and transmission filter 305.In some embodiments, optically filtering layer Transmission filter is combined (for example, coat, be stationed, mechanical couplings) on fibre faceplate can be removed, such as on side.It is removable Except fibre faceplate can provide mechanical support to optical filter, and allow easily to replace light in contact imaging device Learn filter.

Removable fibre faceplate is also utilized to ensure that light is passed with desired angle close to transmission filter and/or imaging Sensor.For example, preferably filtering out exciting light in the embodiment that sample array is imaged using fluorescence.If transmission filter Wave device is interference filter, then this light is preferably close to zero incidence angle close to transmission filter.By in excitation light source Placing between interference filter can be removed fibre faceplate, can reduce incidence angle.Therefore, in some embodiments, transmission filter Wave device is arranged between fixed fibre faceplate and removable fibre faceplate, (as shown in Figure 4) and/or is connect with fixed fibre faceplate Touching.By using the removable fibre faceplate with low numerical aperture or the numerical aperture of the numerical aperture less than fixed fibre faceplate The removable fibre faceplate of diameter, can further decrease incidence angle.In some embodiments, fibre faceplate can be removed has at most The numerical aperture of about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0.In some embodiments, light can be removed The numerical aperture of fine panel be less than fixed fibre faceplate numerical aperture at least about 0.01,0.02,0.05,0.1,0.2,0.3, 0.4, the difference of 0.5,0.6,0.7,0.8,0.9,0.95 or 0.99 unit.In some embodiments, fixed fibre faceplate has extremely Few about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0 numerical aperture.

Normally, the optical fiber in fibre faceplate as disclosed herein be the inner fiber made of a kind of optical material and by Covering made of different materials with different refractivity is constituted.Not the light perfectly aligned with fiber axis inner fiber and covering it Between interface rebound, rest in optical fiber.Correspondingly, by with " black " optical fiber (being alternatively referred to as light absorption optical fiber) generation For some optical fiber in panel, the acceptance angle to optical fiber in panel can be reduced.These or hexagon or circular optical fiber can be with It is spaced apart in the pattern across panel, and shallow angle light can be absorbed, but not interfere the light perpendicular to panel surface.

The fibre faceplate including fixed fibre faceplate and removable fibre faceplate being discussed herein may include absorbability material Material.These materials absorb the light of certain wavelength, and can it is identical as the material found in absorbing filter discussed above or It is similar.For example, absorbent material includes coloured glass and black glass.When being incorporated into fibre faceplate, absorbent material can be with Reduce numerical aperture and/or acceptance angle, so that tighter selecting light based on incidence angle.That is, light must be substantially to hang down Directly in its surface or the angle to form the long axis of optical fiber of fibre faceplate is roughly parallel to close to panel to be transmitted through panel. When adjacent optically filtering layer includes absorbing filter, absorbent material be included in fixed fibre faceplate can be it is useful 's.Such filter can be the fuzzy of image that is thick and leading to sample array.It is coupled to the fixation of absorbing filter Lower numerical aperture in fibre faceplate can partially compensate for this fuzzy.In some embodiments, fixed fibre faceplate In absorbent material also allow to omit individual absorbing filter.

In some embodiments, the core of at least one optical fiber in fibre faceplate or covering include absorbent material (Fig. 5). In some embodiments, the core of all optical fiber in fibre faceplate or covering are absorbabilities.Suction for using in a fiber The property received material can be " black " as defined above.For example, as shown in fig. 6, one or more optical fiber in fibre faceplate or complete The covering of portion's optical fiber can be black.

Substitute the core with black covering and light transmission optical fiber or in addition to the optical fiber with black covering and the core of light transmission it Outside, fibre faceplate can also include the optical fiber of one or more black completely.Black optical fiber can have black covering and black Core, or can be monoblock type, i.e., single refractive index is prepared and/or had by single piece of material.In some embodiments, optical fiber Panel (for example, in fixation fibre faceplate in contact imaging device) includes the multiple black lights being dispersed between multiple optical fiber It is fine.Black optical fiber can be arranged in clearance space between the fibers, as shown in Figure 7.For the ease of manufacture, or in order to make light The acceptance angle of fine panel minimizes, and black optical fiber can have round or hexagon cross section.Any amount of black optical fiber It can be included in fibre faceplate, and these optical fiber can take up any useful part of the cross-sectional area of panel.One In a little embodiments, the quantity of black optical fiber and the ratio of number of optical fiber in fixed fibre faceplate are at most about 1:1000,1: 500,1:200,1:100,1:50,1:20,1:10,1:5,1:2,1:1,2:1,5:1,10:1,20:1,50:1,100:1,200: 1,500:1 or 1000:1.In some embodiments, black optical fiber occupies the cross-sectional area of fixed fibre faceplate at most about 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, 90%, 95% or 99%.In the implementation of statistics Some implementations in, black optical fiber can take up up to about the 0.6% of the fibre faceplate gross area.It will be recognized that working as optical fiber When black optical fiber in panel has greater number, or when occupying the greater portion of cross-sectional area, the acceptance angle of fibre faceplate And/or numerical aperture can be lowered, and the total amount for being transmitted through the light of fibre faceplate can be reduced.

In a further embodiment, there is the fibre faceplate such as institute in Fig. 8 A and Fig. 8 B of one or more completely black optical fiber Show, which replaces and replace the optical fiber of the core with black covering and light transmission.As shown, black optical fiber can have There is the core of the black of monoblock type, wherein in such embodiments, covering can be by the covering with the optical fiber for surrounding fibre faceplate Identical material is made.In other respects, black fibre cladding can have the absorptivity or black similar to black optical fiber Optical fiber can have the diameter for being substantially equal to the combined diameter of the optical fiber with covering.Black optical fiber can be about in fiber facet The various ratios of optical fiber sum in plate are configured, such as with the ratio of a piece black optical fiber of every 61 total optical fiber, such as Shown in Fig. 8 A, or with the ratio of three black optical fiber of every 61 total optical fiber, as shown in Figure 8 B.Further, The quantity of black optical fiber in fibre faceplate may be as few as a black optical fiber of the sum of the optical fiber in fibre faceplate, up to than light Optical fiber sum in fine panel is one few, or any increment of optical fiber in the range.The one of the implementation of statistics In a little implementations, black optical fiber can take up the about 2%-5% of the gross area of fibre faceplate.Further, Fig. 8 C shows optical fiber Panel, has optical fiber light core and covering with black optical fiber combination settings, and black optical fiber is evenly distributed in for fiber facet In total fiber array of plate.

Any one of fibre faceplate discussed above may be provided as optical fiber cone.As fibre faceplate, optical fiber light Cone (also referred to as optical taper or referred to as boring) includes beam of coherent light fibre, but the diameter of optical fiber one end is greater than the diameter of the other end. As a result, two surfaces of the optical fiber cone of optical fiber termination have unequal area, wherein corresponding to the relatively thin of optical fiber The surface at end has lesser area.The light for passing to larger surface from smaller surface is amplified, and is passed to from larger surface smaller The light on surface is lowered.With for example, at least about 1.1:1,1.2:1,1.5:1,2:1,5:1,10:1,20:1,50:1,100:1, The optical fiber of the magnifying power (ratio for being defined as the area of larger surface and the area on smaller surface) of 200:1,500:1 or 1000:1 Light cone can be used in this equipment.In some embodiments of contact imaging device, smaller surface is mechanically coupled into As sensor, which can be less than and make together with standard (non-amplified) fibre faceplate and the sample array of identical size Sensor.In other embodiments, it is small to provide to be mechanically coupled to imaging sensor for the larger surface of optical fiber cone The clearer image of sample array.As desired, optical fiber cone can be mechanically coupled to optically filtering layer discussed above Any layer, and form optical fiber cone optical fiber may include absorbent material.

Presently disclosed equipment may include being embedded in the channel on the surface or inside of one or more layers of equipment.These Channel can accommodate fluid, including interested sample and/or analyte.Therefore, instead of two-dimentional sample array or in addition to two dimension Sample array, sample can be set in the channel for being imaged.In some embodiments, channel has microfluidic dimensions. Channel can be arranged in optically filtering layer, be included in transmission filter and/or removable fibre faceplate.In some implementations In example, channel occur between two layers of equipment interface (for example, between fixed fibre faceplate and optically filtering layer, or Between the removable fibre faceplate and transmission filter of optically filtering layer), or a surface is carved into (for example, from imaging The surface of the farthest optically filtering layer of sensor) in.Channel can be coupled to one or more fluid sources, pump, valve, drainpipe With the other instruments handled for fluid.It will be recognized that the optical device of contact imaging device can be optimized for Sample in channel is imaged.For example, may include there is the fibre faceplate of suitable numerical aperture, or can will absorb Property material is placed in one or more layers of equipment, allows to obtain the focusedimage in channel.This contact imaging device It can be used in combination with slidably equipment to be separated and trace for automatic analyte.In the beauty of commonly assigned co-pending State patent application No.14/642 discloses slidably equipment in 487, and this application is incorporated by reference into this.

Any equipment disclosed herein may include one or more layers with functionalized surface.Such surface can be with Promote the imaging of sample, such as denaturation is contacted without by permission sample and equipment, or allows the layer of equipment (for example, solid Determine fibre faceplate and optically filtering layer) it is slid past each other with the smallest friction.In some embodiments, it is hydrophobic that surface, which is functionalized, Property is hydrophilic.In some embodiments, with the binding partners (for example, antibody) of one or more analytes come functionalization table Face.

E. Image-forming instrument

Some embodiments of presently disclosed equipment are provided in the equipment with lid of such as box etc.Box It may include the shell with substrate and opaque lid.The other embodiments of presently disclosed equipment are provided as not conduct The lid of (outer) shell.Shell is made of durable material (for example, plastics, metal, alloy etc.), and plays and protect the sensor against tide The effect of wet, dirt and other pollutants.Shell is also sensor, fibre faceplate and/or optically filtering layer provide mechanical support, The interruption of Image Acquisition is prevented simultaneously.Substrate may include sensor thermal or the rigid heat-conducting metal plate being coupled to (by Such as aluminium or alloy are made).The plate provides mechanical support for sensor and reduces the temperature gradient across sensor.Lid can be with It is engaged, or otherwise securely closed to ensure that light will not enter during imaging with substrate by retaining ring.Some In embodiment, lid is attached to substrate using one or more hinges.

Box is configured as receiving sample array.In the embodiment that imaging sensor is film, box can also be in light A piece of film is received on the side opposite with sample array of fine panel and optically filtering layer.Substrate and lid may be used as being imaged Sample array is held in place by period, to avoid the fuzzy of gained image.This can be by ensuring lid on substrate Tightly enough closure is not so that sample array and film (if present) move to realize.The box can also include for example existing Substrate inside or the guiding piece on lid, to keep the sample battle array of conventional size (for example, 5 " x7 ", 8 " x10 " or 10 " x12 ") Column.The interior surface of lighttight lid may include flexible or semi-rigid material, such as rubber or foam stuffing or load bullet The pad of spring, to allow some flexibilities of the thickness of sample array.The outer edge of substrate and/or opaque lid can also include Flexible or semi-rigid material (such as rubber edges) is to ensure to be fitted close.

Substrate keeps imaging sensor as described herein, and wherein fibre faceplate and optically filtering layer cover sensor, with For protecting and the signal from sample array being transferred to sensor.Substrate is further configured to the exposure period in imaging sensor Between or signal (e.g., including the data of digital picture) is transferred to external equipment (for example, processor, scanner, calculating later Machine).The control panel for being coupled to sensor can be used for transmitting the signal from sensor, and is communicated to and optionally has The external equipment of user interface.External equipment may include touch screen (for example, integrated in the lid perhaps separated) or It can be handheld device (for example, tablet computer or smart phone) either other processors or computer.Communication can be with By the cable or port (for example, USB or Ethernet) on box or wirelessly (for example, using bluetooth or other wireless transmissions) To occur.

Presently described equipment can be provided with any convenient size.Typical Western blotting, point trace, Northern trace and Southern trace are several inches of magnitudes (such as 2 inches × 3 inches, 3 inches × 4 English in every side It is very little, 4 inches × 8 inches), so that relatively small box and sensor detection zone will be enough for most of measurements, such as In the range of about 10 inches × 12 inches.Sequencing gel is usually biggish, thus can provide with larger size (such as In 20 inches × 36 inch ranges) box.

It in some embodiments, include label, such as LED (light emitting diode), LCD (liquid in the inside of box or shell Crystal display) or compact illumination display.Label can be used to mark the image generated with imaging sensor, or by sample battle array Column are aligned with imaging sensor.For example, compact LED or back-illuminated type LCD can be placed on the angle of lid, substrate or fibre optic plate It falls, to mark the designated position being used as on a piece of film of imaging sensor.LED or LCD can be opened in short cycle to keep away Exempt from that film is made to be saturated (for example, -2 seconds 0.1 second, the comparison intensity depending on the signal from sample).It is passed comprising digital imagery In the embodiment of the equipment of sensor, LED or LCD signal can be for example adjusted using non-Destructive readout (NDR) imager Intensity.In some embodiments, LED or LCD are configured in signature, for example, user name initial, date etc., and can To be substituted as or be programmed.For example, LCD can have back illumination to allow through programming quartz crystal device to allow light with certain Kind pattern is by being signature, character or symbol by signal shaping.LED may include lattice array or can be included in LED In array.

F. measurement and material

Presently disclosed equipment is suitable for dependent on fluorescence, chemiluminescent, bioluminescence or radioactive letter Number measurement.Such measurement includes Western blotting, ELISA, immunoassays, Northern trace (for example, using report base The expression study of cause), Southern trace, nucleic acid sequencing measurement and be related to virus or bacterium label measurement (for example, CFU or Expression study).Measuring agreement can be in for example fertile gram (Walker) (2009 third edition) protein protocol manual (Protein Protocols Handbook)；Henrique Hilario (Hilario), by the agreement of the foranalysis of nucleic acids of nonradioactive probe (Protocols for Nucleic Acid Analysis by Nonradioactive Probes) (2007 second edition)；Temperature Gram (Wenk), biochemistry protocols handbook (A Manual for Biochemistry Protocols) (2007)；Harris (Harris), it is found in cell biology agreement (Cell Biology Protocols) (2006) etc..

In some embodiments, measurement be related to will likely include target analytes (for example, protein, DNA or RNA) sample Product are transferred to film (such as trace)；And expose the film to be specifically bound to target analytes the first binding reagents (for example, through The probe or antibody of label).Therefore, film may serve as the two-dimensional array for analyzing analyte detection.Before transfer, sample can With on gel (for example, running gel of such as PAGE or Ago-Gel etc) or culture dish, or can be by user It is applied directly on film.As discussed further below, in some embodiments, measurement is special further to exposing the film to Property combine the first binding reagents the second binding reagents (for example, labeled secondary antibody or labeled such as Streptavidin it The affinity agent (combine biotin on the first binding reagents) of class).Such measurement typically relates to combining between step Washing.

In some embodiments, sample is applied directly to film or other arrays, such as is moved on film with pipette.This can To help to control (such as dilution series of the known quantity with analyte) or positioning signal.

Once any convenient technology, which on two-dimensional arrays or in two-dimensional array, can be used, in distribution detects sample according to expectation The analyte of product.In some embodiments, if interested analyte includes detectable label or connects with this label It connects or is coupled, then can detect interested analyte on array.The example of detectable label includes chromophore, fluorescence Group and radioactive isotope.In the case where no label, analyte can also be detected directly, if they are that optics is living If property.For example, protein and nucleic acid absorb infrared ray and ultraviolet radiation, and fluorescence can also be shown.It correspondingly, can be with These analytes are detected by the way that the light of appropriate wavelength is directed on array and measures the interaction between light and analyte. For the protein analyte containing trp residue, by analyte being had with what several halogens replaced under uv radiation depositing The contact of any one of machine compound (such as chloroform, 2,2,2- ethapons or 2,2,2- trichloroacetic acids), can be enhanced glimmering Light.Such as in United States Patent (USP) No.7,569,130 and 8,007,646 and other places described in, in such a situa-tion, in tryptophan The reaction of UV photoinduction occurs between the organic compound that indolyl moiety and halogen replace, so as to cause sending out at visible wavelengths The fluorescent chemicals penetrated.

Any label with analyte direct or indirect connection, such as United States Patent (USP) can be used in the detection of analyte No.6,165,800,6,395,503,6,972,326 and 7, those of described in 935,489.In some embodiments, it detects Label be fluorescence.It can include fluorescein, rhodamine, cumarin, BODIPY and cyanine with the fluorescent dye marked.Other Fluorescent dye can be used and for example in Johnson (Johnson) and Si Pengsi (Spence) (eds.), Molecular Probes Handbook-A Guide to Fluorescent Probes and Labeling Technologies (molecule Probe handbook-fluorescence probe and labelling technique guide) (the 11st edition), Eugene city (Eugene), Oregon (Oregon): It is summarized in 2010.Enzyme addition (enzymatic addition), click chemistry (click chemistry) can be used or apply Tao Dingge connection (Staudinger ligation) is on demand by conjugated fluorescent dyes to analyte.In addition to organic dyestuff, quantum dot (" Q- point ") and fluorescent polymer nano particle (polymerization object point or " P- point ") are also used as fluorescent marker.With any big The quantum dot of small, color or ingredient can be used, and can be produced according to expectation and be coupled (method survey with analyte In for example " Medintz etc., Nature Materials (nature material) 4:435-446,2005 ").Similarly, any polymer Point (such as " Wu and Chiu, Angewandte Chemie 52:3086-3109,2013 " and elsewhere it is described that It can be coupled to analyte a bit) for detecting.Fluorescence can also be by being attached to fluorescin (such as these analytes Can be used as the green fluorescent protein (GFP) or yellow fluorescence protein (YFP) of label) it is endowed analyte.It is recombinantly expressing In system, fluorescin can be synthesized to the part of same polypeptide together with protein analyte so that fluorescin and point Analysis object is covalently linked together, and one keeps another detectable.

In some embodiments, object is tested and analyzed using chemiluminescence.These embodiments are related to experience chemical reaction simultaneously Luminous is usually the chemiluminescent substrate of small molecule.Some reactions of chemiluminescent substrate can be by enzymatic.For example, Rumi Promise is aoxidized by Catalyzed Synthesis By Peroxidase.Various phosphorylations 1, the luminous decomposition of 2- dioxetane is by phosphatase catalytic, and half The decomposition of the sugar-substituted 1,2- dioxetane of cream is by galactoside enzymatic.As used in the tyramide signal amplifying technique Tyrasamine derivative can be converted into tyrosine reactivity free radical by peroxidase.Other common chemiluminescent substrates and mark Note includes NADP, umbelliferone phosphate and p- nitrophenyl phosphate.It include horseradish peroxidating for the enzyme in chemical luminescent detecting Object enzyme (HRP), alkaline phosphatase, beta-D-galactosidase, glucose-6-phosphate dehydrogenase (G6PD) and xanthine oxidase are (referring to example Such as the Krick of background (1991) Clin Chem 37:1472).Any system or known in the art in these systems Other systems, which can be used by, is coupled to analyte for substrate or enzyme to detect interested analyte.Or substrate therefore Or enzyme can be by the detectable label as analyte.When substrate and enzyme are contacted, light emitting and analyte common location.

Chemiluminescence system (i.e. bio-luminescence system) from living organism can also be used in analysis analyte detection.Example Such as, luciferin, which can be coupled to analyte and work as, is exposed to luciferase (for example, Fluc) or jellyfish hair It is detected when photoprotein matter.Preferably, for the purpose of detection, chemiluminescent substrate or for the enzyme and analyte of the substrate It is any to couple the reaction for not interfering substrate.In some embodiments, enzyme used in chemiluminescence detection is affine by biotin- Element connection is coupled to interested analyte.For example, one or more polypeptides of enzyme can be covalently linked to Avidin, And analyte can be biotinylated.Therefore, enzyme and analyte due between biotin and Avidin unit in conjunction with and become It must be connected.

In other embodiments, testing and analyzing object includes making sample array and the one or more analytes for being directed to sample Binding partners contact, and detect the signal for indicating the combination of the binding partners and one or more analytes.Such detection quilt For in electroblotting method (such as Southern blotting, Northern blotting and immunoblotting), and electric print can be used Detection reagent used in mark method and instrument.Binding partners may include antibody, enzyme, protein (such as Avidin or strepto- it is affine Element), it is peptide, aptamer, ligand, nucleic acid (such as nucleotide or oligonucleotides), nucleic acid analog, coordination complex, natural or synthetic Polymer, carbohydrate or small molecule (such as biotin).Specifically, when the analyte in sample is protein, knot Closing companion can be antibody.The antibody can be for the epitope in one or more analytes of interest analytes.Antibody can be directly It is detectable, such as by carrying fluorescent marker, or secondary antibody and/or chemiluminescence, which can be used, be detected antibody can.Work as sense When interest analysis object is nucleic acid, one or more binding partners can be the complementary nucleic acid sequence with fluorescence or radioactively labelled substance Column.Other probes for all kinds of analytes are known, and the signal that detectable multiclass instruction combines.In some embodiments In, signal includes chemiluminescence, electroluminescent, fluorescence, infra-red radiation, radioactivity, color or light absorption.In general, it analyzes Object and binding partners can be the part of bio-sensor system, which can be used additional molecular components or detecting instrument.

Signal caused by the combination between any suitable technology amplification analyte companion in connection can be used.Example Such as, when using one or more antibody test analytes, tyrasamine group amplified signal can be used.Ortho position connection also can be used to survey Surely carry out amplified signal, the antibody of two different oligonucleotides connections is by common location in the measurement, and thus oligonucleotides can quilt It connects and amplifies.It instead or in addition, can be by one or more detectable labels, such as fluorogen, polymerization object point or quantum Point is coupled to increase signal, all those signals as mentioned before with analyte and/or binding partners.For example, coupling can utilize life Object element-Avidin interaction.If fluorogen is coupled to each of analyte and binding partners, and both fluorogens Fluorescent quenching or fluorescence resonance energy transfer (FRET) can be used then to detect combination in excitation and emission spectra with overlapping.? In some embodiments, make additive, such as crowded dose (such as polyethylene glycol or glucan) contact sample array in the detection process To improve the association rate between analyte companion in connection.

If desired, it can while or be detected on same sample array in different moments using two or more binding partners Analyte.Binding partners can be specific to same analyte, the different form of same analyte (such as phosphorylation and non-phosphoric acid Change) or entirely different analyte.These binding partners can produce identical signal, can geodetic unlike signal (such as with not With the fluorescence of launch wavelength) or uncorrelated type signal (such as fluorescence and radioactivity).It can be mentioned using multiple binding partners For the analysis analyte detection of more information more possible than single binding partners.For example, two kinds of binding partners can disclose and be fixed on battle array Relative position of the relative quantity or analyte of two kinds of different analytes on column on array.Alternatively, for same analysis Presence, integrality or the accessibility of detectable two kinds of two kinds of different antibodies of object different epitopes.

In some embodiments, the analyte on test sample array can be needed the binding partners exposure for analyte In reagent.The reagent is in combination with binding partners or reacts with it to generate detectable signal.For example, if analyte is albumen Matter and binding partners are antibody, then the reagent can be chemiluminescent substrate (such as luminol), which can be coupled in this The enzyme domains (such as horseradish peroxidase) of antibody aoxidize.Substrate can be added thereto in the solution for being soaked with array, not with Analyte or antibody conjugate, but chemiluminescence signal discloses the position of the analyte in conjunction with antibody.In order to amplify substrate oxygen Change the light of transmitting and obtain the chemiluminescence of enhancing, chemical substance can also be added, such as to iodophenol.Alternatively, when will resist When body is used as the binding partners for analyte, the reagent for testing goal is alternatively the secondary antibody of tape label.It will recognize that The plurality of reagents in addition to binding partners also can be used to detection.

In some cases, the analyte detected on array can be related to using sealer.Sealer can be tied non-specifically Combined array column, such as it is incorporated into the position of unlocked analyte, prevent the binding partners for analyte from also non-specifically combining In these positions.Sealer can reduce background signal as a result, allow the detection of analyte more accurate.The example of sealer includes Protein, such as bovine serum albumin(BSA) or lactoprotein.Preferably, before making array contact binding partners, by sealer application In array.

It if desired, can (such as protein, nucleic acid or other analytes have been shifted to by the array for carrying sample Film) it is exposed to the fluid of index matching.The fluid of index matching increases the transparency of array, and/or matching in equipment In fibre faceplate or sensor refractive index.Therefore, the fluid of index matching prevents array itself from upsetting point in array Analyse the optical signalling that object generates.For example, fluid can reduce the reflection of the interface between array and fibre faceplate.Refractive index The example for the fluid matched is the fused silica matching fluid from Cargille.

In some embodiments, measurement is related to labelled with radioisotope, such as transmitting gamma-rays, positive electron, β and α The radionuclide of son and X-ray.Suitable radionuclide includes but is not limited to²²⁵Ac、⁷²As、²¹¹At、¹¹B、¹²⁸Ba、²¹²Bi、⁷⁵Br、⁷⁷Br、¹⁴C、¹⁰⁹Cd、⁶²Cu、⁶⁴Cu、⁶⁷Cu、¹⁸F、⁶⁷Ga、⁶⁸Ga、³H、¹⁶⁶Ho、¹²³I、¹²⁴I、¹²⁵I、¹³⁰I、¹³¹I、¹¹¹In、¹⁷⁷Lu、¹³N、¹⁵O、³²P、³³P、²¹²Pb、¹⁰³Pd、¹⁸⁶Re、¹⁸⁸Re、⁴⁷Sc、¹⁵³Sm、⁸⁹Sr、^99mTc、⁸⁸Y and⁹⁰Y.Certain In embodiment, radioreagent may include¹¹¹In-DTPA、^99mTc(CO)₃-DTPA、^99mTc(CO)₃-ENPy2、^62/64/67Cu- TETA、^99mTc(CO)₃- IDA and^99mTc(CO)₃Triamine (cyclic annular or straight chain).In other embodiments, reagent may include DOTA With its with¹¹¹In、¹⁷⁷Lu、¹⁵³Sm、^88/90Y、^62/64/67Cu or^67/68The various analogs of Ga.

G. system

It according to the system in the embodiment of the present invention include contact imaging device as described above and light source (such as exciting light Source).Light source can be used for irradiating the sample or sample array for being placed as contacting with the equipment for imaging.The example packet of light source Include incandescent lamp, fluorescent lamp, light emitting diode (LED) and laser.In some embodiments, wavelength (example of the light source across wide scope Such as, about 50nm, 100nm, 200nm, 300nm, 400nm or 500nm) transmitting, and in other embodiments, transmitting is limited in narrow Wavelength band (for example, about 5nm, 10nm, 20nm, 50nm, 100nm or 200nm) or single wavelength.If desired, light source can be quasi- Directly.It may be used in this system in the light source that ultraviolet, the visible and/or infrared part of electromagnetic spectrum emits.Light source can be with Optionally it is chosen for one or more of measurement as described above.For example, light source can emit the wavelength of light to excite Fluorescent marker in interested analyte.

The system may further include fluorescence, phosphorescence or chemiluminescence sample.Sample may include it is one or more can With the substance or analyte being detected optically.Sample can be arranged on two-dimentional sample array (such as electrophoresis as described above Gel or blotting membrane) in, which can be placed proximate contact imaging device.It alternatively, can be by sample It is applied directly to contact imaging device, is such as applied to the surface of the exposure of optically filtering layer.The embodiment of system can be by It is configured so that the light emitted by excitation light source passes through sample.For example, exciting light can be perpendicular to the plane of two-dimentional sample array Or it advances with the plane at low-angle, and pass through sample array before reaching contact imaging device.

In some embodiments, which includes excitation fiber panel, which is configured as setting and is swashing Between light emitting source and sample.Excitation fiber panel can with any fibre faceplate discussed above (for example, fixed fibre faceplate, Fibre faceplate or optical fiber cone can be removed) characteristic is shared, and the light from light source is directed to sample.Excitation fiber panel is therefore It can be used for controlling the geometry for reaching the light of sample and imaging sensor.It can be built between light source and excitation fiber panel Any distance is found, and if desired, excitation fiber panel can be mechanically coupled to light source.

In some embodiments, excitation fiber panel has low numerical aperture, and only transmission is approximately perpendicular to panel The light on surface.For example, numerical aperture can be at most about 0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0.If excitation fiber panel is roughly parallel to the optically filtering layer of contact imaging equipment, light reaches sample with low incidence angle Plane and optically filtering layer (Fig. 9 A and 9B).Correspondingly, optically filtering layer can such as by being designed in conjunction with interference filter, To prevent exciting light from passing through and reach imaging sensor.In some embodiments, since light source itself is collimated, and/or due to Excitation fiber panel is only by the light with small incidence angle, so being emitted by light source and by the light quilt of excitation fiber plane transport Collimation.(Fig. 9 C) in a further embodiment can be used what TIR structure was uniformly diffused towards sample and fibre faceplate transmitting Light.

As the surface for being placed on contact imaging device light source (for example, wherein optical fiber with optically filtering layer The vertical angle in surface reach optically filtering layer) alternative solution, light source can be placed on to the side of contact imaging device Face (Figure 10).Therefore, light enters sample array from side, and hits the most or all of light of optically filtering layer by from sample The reflection of analyte in product array or fluorescent emission generate.In this configuration, the geometry of system prevents exciting light from arriving Up to imaging sensor.In some embodiments, excitation light source is positioned relative to contact imaging device, so that by excitation light source The light of transmitting is not incident on optically filtering layer.For example, the sample that excitation light source can be placed on contact imaging device is flat The beside and under in face, therefore the light from light source is not directly fallen on optically filtering layer.In some embodiments, it excites Light source is positioned relative to contact imaging device so that by the light of excitation light source transmitting with from normal at least about 1,2,3,4,5, 6,7,8,9,10,20,30,40,50,60,70,80 or 90 degree of angle is incident on optically filtering layer.In some embodiments, Optically filtering layer can be in conjunction with absorbing filter to remove the exciting light close with high incidence angle.Light source can also be positioned as making Exciting light is obtained to be parallel to the direction on the surface of optically filtering layer and advance.For example, excitation light source can be laser, wherein light beam Surface across optically filtering layer is guided or is guided from the side of optically filtering layer but does not strike against surface.Further implementing In example, optically filtering layer be can be with reflecting material, light source or by optical filter back-illuminated (Fig. 9 C).

H. method

Equipment and system disclosed above can be used to carry out the contact of fluorescence, phosphorescence or chemiluminescence sample at Picture.In the method, sample is disposed close to contact imaging device.For example, two-dimentional sample array is placed on equipment On optically filtering layer, or sample is otherwise applied to the layer as needed.Then excitation light source is exposed the samples to, And the image of sample is obtained using the imaging sensor of contact imaging device.

Any exciting light in excitation light source discussed above (for example, LED or laser) can be used in the method Source.Light source can be positioned in above sample, sample side or elsewhere, and can according to need and activated.Equally Ground, imaging sensor, which can according to need, to be activated, such as is caused in the case where CCD or cmos sensor using external control panel It is dynamic.In some embodiments, light source and imaging sensor are under the control of common circuit or software, with suitably time exposure And Image Acquisition, and/or ensure using the light of appropriate amount to form image.It can according to need setting contact imaging device week The ambient light levels enclosed, such as by the way that equipment is placed in dark room or equipment is enclosed in lighttight box (discussed above).

In some embodiments, the image of sample is obtained while sample is exposed to excitation light source.This can work as glimmering Light analyte is completed when being just detected, and image is formed by the light re-emitted in excitation from analyte fluorescence.Work as sample In product when the fluorescence lifetime of analyte short (millisecond magnitude or smaller), while being imaged and being exposed to exciting light and can be necessary. This service life to obtain image after light source has been off to be unpractical.In these embodiments, contact at As the fixation fibre faceplate and optically filtering layer of equipment can be designed as described above to prevent exciting light from reaching imaging sensor. Similarly, excitation light source can be positioned or be otherwise configured to (for example, being placed on behind excitation fiber panel), with Minimize exposure of the imaging sensor to exciting light.

In other embodiments, the image of sample is obtained using time-resolved fluorescent technique.It is, for example, possible to use have The fluorogen of long-life excitation state；Pulse laser with optical gate control；Quickly detection electronic device；And/or have nanosecond or Picosecond the imaging sensor of temporal resolution obtain image.In these embodiments, it can have been closed in excitation light source Image is formed by the light from fluorescent emitted after closing.This method can be similarly effected for phosphorescence sample, the phosphorescence Sample can be shone after the exposure to the exciting light up to many seconds or many minutes.It will recognize that being exposed in the sequence the step of Light and Imaged samples provide the ability being imaged in the case where excitation light source is closed to the configuration of this equipment and system Greater flexibility.For example, fixed fibre faceplate and optically filtering layer do not need to be designed to stop exciting light, and exciting light Source can be placed on the place of most convenient, without being located in its place that will not be incident on imaging sensor.

Image Sharpening Algorithm can also be applied to the image obtained using contact imaging device by this method.These algorithms The reduced spatial resolution in image can be wholly or partly compensated, this may be due to the bad source figure from panel As, dislocation or the received image in nonideal position, the accidentally sample or sensor of calibration or fuzzy and generate from sensor. The fuzzy sense that can reach imaging sensor by passing through thick sample array (Figure 11), optical filter or other non-light guide materials again Light that the analyte of interest issues and generate.The Image Sharpening Algorithm of standard can be used, such as utilize Airy disk point spread function Number (Airy disc point-spread function) deconvolutes.

I. external member

The embodiment of the present invention is also provided as to fluorescence, chemiluminescence or the phosphorescence sample in contact imaging configuration The external member of product imaging.As discussed under " equipment " above, external member includes being mechanical coupling to the imaging sensing of fixed fibre faceplate Device.External member further includes two or more optically filtering layers (for example, the first optically filtering layer and second optically filtering layer), can To be substituted for each other in contact imaging device.Each optically filtering layer includes that fibre faceplate and optical filter can be removed simultaneously Be configured as being mechanical coupling to fixed fibre faceplate, can be removed fibre faceplate side with optical filter in conjunction with (such as coat, It is stationed, mechanical couplings).Optically filtering layer can change to and swap out as needed contact imaging device with to different samples at Picture, or identical sample is imaged in a different configuration.

In some embodiments, the wave-length coverage that the optically filtering layer in external member is passed through or filtered out by them is by each other It distinguishes.For example, an optically filtering layer may include short logical optical filter, and another includes long logical optical filter.Light The light of different wave length section can also be passed through by learning wave filtering layer.For example, the first optically filtering layer can lead to using band logical optical filter It crosses wavelength visible (for example, feux rouges), and the second optically filtering layer can pass through short-wavelength visible light (for example, blue light).This Two kinds of optically filtering layers can stop the visible light (such as green light) of medium wavelength, infrared light and/or ultraviolet light.In these implementations In example, the selection of optically filtering layer for particular assay can wavelength based on the light emitted by analyte in sample or by with In the wavelength of the light of excitation analyte.

Optically filtering layer in external member can pass through the additional of transmission filter in every layer and/or removable fibre faceplate Characteristic is distinguished.In some embodiments, the transmission filter of the first optically filtering layer is delectric filter, and the second optics The transmission filter of wave filtering layer is absorbing filter.As discussed above, dielectric filter and absorbing filter filter not at them It is different in terms of efficiency with the light under incidence angle.Similarly, fibre faceplate can be removed to depend on numerical aperture and permit different Light under incidence angle.In some embodiments, the removable fibre faceplate of the first optically filtering layer and the second optically filtering layer has There is different numerical apertures.Pass through the optically filtering layer including effectively filtering or passing through the light within the scope of different angle, external member Different from different types of sample array (for example, thick or thin) and light source can configure (such as on sample array Side or side) it is used together.

Optically filtering layer in external member can also be by can be removed fibre faceplate and transmission filter relative to fixed optical fiber The expected orientation of panel is to distinguish.In some embodiments, the first optically filtering layer is configured as setting removable fibre faceplate It sets between fixed fibre faceplate and optical filter, and the second optically filtering layer is configured as transmission filter being arranged Between fixed fibre faceplate and removable fibre faceplate.That is, optically filtering layer can be configured to by or removable optical fiber Panel or transmission filter are set as adjacent with fixed fibre faceplate, and external member may include for both configuration optimizations Optically filtering layer.

As discussed above, removable fibre faceplate is positioned to contact with fixed fibre faceplate allows light to be conducted through this Two panels are without losing, such as from transmission filter to imaging sensor.This configuration can be contact imaging device And/or sample provides increased mechanical support.Alternatively, transmission filter is positioned to contact with fixed fibre faceplate, is made Fibre faceplate must can be removed towards sample, the light from sample is allowed to be primarily based on incidence angle quilt between filtering based on wavelength Filtering.For any orientation, the removable fibre faceplate and transmission filter of optically filtering layer can be selected consistently so that suitable When wavelength and incidence angle light by for imaging task on hand.In some embodiments, one in external member or more A optically filtering layer is reversible, because of the either side (present and fibre faceplate or optical filter can be removed) of optically filtering layer Fixed fibre faceplate can be mechanically coupled to.

In the appended claims, term "a" or "an" is intended to indicate that " one or more ".It is walked when in narration When before rapid or element, term " including (comprise) " and its such as " including (comprises) " and " including Etc (comprising) " deformation is intended to mean that the addition of further step or element is optional and is not arranged It removes.

All references recited herein (for example, patent, patent application, books, journal of writings or other publications) pass through It is whole to quote and be incorporated into for all mesh, as each independent document is by particularly and individually for all purposes by drawing It is incorporated to entire contents.These documents being incorporated by reference into include the case where disclosure in the description contradicts Under, specification is intended to replace and/or prior to any conflicting material.

As will be it would be apparent to those skilled in the art that the case where not departing from the spirit and scope of the present invention Under, many modification and variation can be carried out to the present invention.If for example, any aspect described above can be combined into one or Dry different configuration, each configuration have the subset of aspect.Further, in the description of entire front, for the mesh of explanation , elaborate several specific details in order to provide thorough understanding of the present invention.Particular embodiment described herein, which is only used as, to be shown Example provides, and is not meant to be limited in any way.Description and embodiments are intended to be regarded only as to be exemplary, this The true scope and spirit of invention are indicated by the appended claims.

Claims (20)

1. a kind of contact imaging device, comprising:

Imaging sensor；

Fixed fibre faceplate, is mechanically coupled to the imaging sensor；With

Optically filtering layer is mechanically coupled to the fixed fibre faceplate.

2. contact imaging device according to claim 1, wherein the optically filtering layer includes interference filter, wherein The interference filter is configured as long pass filter, with block ultraviolet, or make visible light by or combinations thereof.

3. contact imaging device according to claim 1, wherein the optically filtering layer includes absorbing filter.

4. contact imaging device according to claim 2, wherein the absorbing filter is arranged on the interference filter Between wave device and the fixed fibre faceplate.

5. contact imaging device according to claim 1, wherein the fixed fibre faceplate includes multiple optical fiber, and The core or covering of at least one optical fiber in the optical fiber include absorbent material.

6. contact imaging device according to claim 1, in which:

The optically filtering layer further comprises that fibre faceplate and transmission filter can be removed,

The removable fibre faceplate in side by conjunction with the transmission filter, and

The optically filtering layer is configured as removing from the fixed fibre faceplate.

7. contact imaging device according to claim 6, wherein the transmission filter is interference filter or absorption Filter.

8. contact imaging device according to claim 6, wherein the numerical aperture of the removable fibre faceplate is less than The numerical aperture of the fixed fibre faceplate.

9. contact imaging device according to claim 6, wherein the removable fibre faceplate be arranged on it is described solid Determine between fibre faceplate and the transmission filter, or wherein the transmission filter is arranged on the fixed fibre faceplate Between the removable fibre faceplate.

10. contact imaging device according to claim 1, wherein the fixed fibre faceplate is optical fiber cone.

11. contact imaging device according to claim 1, wherein the acceptance angle of the optical fiber of the fixed fibre faceplate is Most about 30 degree.

12. contact imaging device according to claim 1, wherein the fixed fibre faceplate includes being dispersed in multiple light Multiple black optical fiber among fibre, wherein the multiple black optical fiber is arranged in the clearance space between the optical fiber, place The quantity of the black optical fiber of Yu Congyue 1:61 to about 60:61 and the ratio of number of optical fiber, or occupy the fixed fibre faceplate About the 10% of cross-sectional area.

13. contact imaging device according to claim 12, wherein the black light fibre have circular cross section or The cross section of hexagon.

14. a kind of system for fluorescence, phosphorescence or chemiluminescence sample to be imaged, the system comprises excitation light source and Contact imaging device, the contact imaging device include:

Imaging sensor；

Fixed fibre faceplate, is mechanically coupled to the imaging sensor；With

Optically filtering layer is mechanically coupled to the fixed fibre faceplate.

15. system according to claim 14 further comprises box, wherein the box includes substrate and lighttight Lid, and the contact imaging device is mechanically coupled to the substrate, wherein the substrate includes that heat-conducting metal is flat The plate and imaging sensor is coupled to the metal plate.

16. system according to claim 14 further comprises excitation fiber panel, wherein the excitation fiber panel quilt It is configured to be arranged between the excitation light source and the sample, and at most about 1.0 numerical aperture.

17. system according to claim 16, wherein being emitted by the excitation light source and by the excitation fiber panel The light of transmission is collimated.

18. system according to claim 14, wherein the excitation light source is determined relative to the contact imaging device Position is not at being incident on the light emitted by the excitation light source on the optically filtering layer.

19. system according to claim 14, wherein the excitation light source is determined relative to the contact imaging device Position is at being incident on the light emitted by the excitation light source on the optically filtering layer with the angle away from least about 30 degree of normal.

20. system according to claim 14, wherein the excitation light source is determined relative to the contact imaging device Position is at making excitation light source emit light on the direction on surface for being parallel to the optically filtering layer.